Herpes simplex viruses 1 and 2 (HSV-1, HSV-2) cause a wide range of serious diseases, including oral/genital lesions, corneal blindness and eczema herpeticum. HSV-1 and HSV-2 are both transmitted sexually and can infect babies during birth. Therapy for HSV relies primarily on nucleoside analogs such as acyclovir (ACV) which are insufficiently effective, and resistant viruses are becoming common among immunosuppressed persons. Therefore, there is an urgent need for better anti-herpesvirus drugs. The nucleotidyl transferase superfamily (NTS) of enzymes comprises a range of nucleases and recombinases that are essential for DNA replication, recombination, and nucleic acid turnover. Our screening program to identify inhibitors of HSV replication demonstrated profound activity in vitro by two NTS enzyme inhibitors, ciclopirox and piroctone, which are approved drugs for other indications. These two drugs also suppress replication of ACV-resistant HSV strains. In this STTR project, we will execute a program to translate the observed anti-HSV in vitro of these two select drug candidates into topical application in an animal model of HSV-2 disease. Additionally, the potent action of these drug candidates in vitro will be examined in combination with ACV to evaluate synergy. Aim 1: Execute a proof-of-concept in vivo demonstration of the anti-HSV activity of formulated drug candidates for topical application in a mouse model of HSV-2 infection. We anticipate topical treatment with formulated ciclopirox and piroctone will suppress HSV-2 replication in the genital tract and the nervous system, and reduce genital disease and weight loss without inducing unacceptable toxicity. The experiments will yield critical preliminary data about the effectiveness of topical treatment with these drugs for suppression of HSV-2 replication and disease, and a guide for the effective dose. Demonstration that either or both of these formulated drug candidates reduces HSV-2 replication and spread in vivo would strongly support development of these NTS enzyme inhibitors as drugs for herpesvirus infections. Aim 2: Evaluate the potential for synergy and toxicity between ciclopirox and/or piroctone and acyclovir in vitro. We have strong data indicating ACV and ciclopirox and piroctone have different mechanisms of action, and preliminary data showing in vitro synergy between ciclopirox and ACV. Therefore, we anticipate synergism will be confirmed in vitro for either or both of the select drug candidates and ACV. Significant Impact: New therapies are urgently needed for patients who suffer from recurrent outbreaks of HSV, and especially immunocompromised individuals infected with nucleoside analog-resistant strains. This project will translate in vitro results into an animal model of disease and explore synergy of drug combination(s). The goal is to guide development of new therapeutic drug candidate(s) for treatment of herpes-virus infections, including HSV-2, as monotherapies or in synergistic combination with existing drugs.